Cryptococcus neoformans is one of the most common life-threatening central nervous system infections in humans and despite present treatments, morbidity and mortality still remain high. In a recent outbreak on Vancouver Island, British Columbia it was shown that cryptococcal strains have evolved rapidly to produce more virulent strains. The focus of this proposal is to use molecular techniques to identify, characterize, and validate genes in C. neoformans which are important to the virulence composite of this encapsulated yeast. Our primary hypothesis is based around the simple assumption that under certain environmental stresses, C. neoformans strains will regulate their genetic networks/pathways for production of proteins to allow them to survive and grow in the hostile environment of the host. This proposal details a plan of investigations which allows use of new technological advances to perform a highly integrative but global screen of transcriptional profiling in order to predict the weak points in Cryptococcus pathobiology. Our laboratory has over the last few years validated that this overall strategy can be successfully used. We have used cDNA subtraction techniques, differential display RT-PCR, in vivo expression technology, serial analysis of gene expression (SAGE), and most recently microarray technology to successfully begin to identify genes and which make C. neoformans, a pathogen. Through our studies we have identified the following genes and pathways to be important to the virulence composite: 1) genes encoding enzymes; 2) oxidative stress genes; 3) signaling pathway genes; 4) high temperature growth genes; 5) impact of mitochondria and respiration; 6) influence of attenuated null mutants on host immunity. A cornerstone of this proposal is to use microarrays to harness their ability to collect massive data and yet attempt to focus our understanding to relevant genes and networks by using specific in vitro and vivo conditions. This screening design will be carefully linked to functional studies through creation of null mutants for phenotypic analysis in relevant animal models. Our overall scientific plan is to understand the genetic regulation of the virulence composite which will contain a powerful insight into discovery of gene targets that interrupt pathogenesis and lead to new therapeutic strategies such as antifungal drugs and vaccines. PROJECT NARRATIVE This project uses genomic strategies to capture what makes the life-threatening fungus Cryptococcus neoformans such an effective pathogen in immunocompromised individuals. With the knowledge gained from these studies it is anticipated that new antifungal targets can be identified to help develop new drugs and/or vaccines against this common fungal pathogen for an enlarging severely immunocompromised population.